Abstract
Purpose :
The Humanin (HN) peptide was the first identified mitochondrial derived peptide (MDP), is 24 amino acids long and has neuroprotective and anti-apoptotic properties. In rodent animal models and human clinical studies, it has been reported that aging and age-related diseases are correlated with the decreasing levels of serum HN.
Our preliminary study showed that endogenously synthesized humanin peptide degraded in human plasma samples. We found humanin fragments as well as their oxidized and dimerized forms in human plasma samples.
In this study, we analyzed human plasma samples using ultra-performance liquid chromatography coupled to high resolution mass spectrometry (UPLC-HRMS) and identified various humanin fragments, their oxidized and dimerized forms in human plasma samples.
Methods :
Older normal subjects (n=5) provided plasma samples which were frozen until use. Briefly, plasma samples were thawed and diluted to 20% ethyl nitrile. Diluted plasma samples were filtered using ultrafiltration devices (Amicon Ultra-4 10kDa) at 17000 g for 30 mins at 4oC. After filtering, the samples were diluted 10 times with LC-MS grade water. The final solutions were analyzed immediately using by UPLC-HRMS to determine the degradation products and oxidized products of the humanin peptide in these plasma samples. The data collected were further analyzed using the BiopharmaLynxTM software to identify the specific amino acids within each HN fragments.
Results :
Our preliminary study showed that endogenously synthesized humanin peptide degrades in human plasma samples. We found 16 different humanin fragments and 3 oxidize forms of humanin fragments in human plasma samples.
Conclusions :
Degraded humanin fragments in human plasma samples have been analyzed using advanced UPLC-HRMS technologies as well as identified humanin fragments and their intensity levels using BiopharmaLynxTM software. The critical role of humanin fragments in physiological conditions and human diseases are targets for future studies as well whether there are any biological activities of the humanin fragments. Our results may help in developing better experimental methods for cell culture and animal studies to further understand the importance of degradation products of humanin in diseases.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.